摘要目的 研究广州地区先天性感染的人巨细胞病毒(HCMV)临床低传代分离病毒株UL144基因序列的多态性,探讨UL144基因在HCMV致病中的作用.方法 对3株经多重PCR鉴定HCMV DNA为阳性的临床低传代分离株进行HCMV UL144基因全序列PCR扩增,PCR产物克隆到pMD18-T载体上再测序,将其序列与GenBank中公布的其它10株临床分离株UL144基因一起进行分析.结果 本实验克隆并测序了HCMV临床低传代D3、D2和D52病毒株的UL144基因,提交GenBank,已被GenBank收录,序列号分别为DQ180368、DQ180382和DQ180355.HCMV临床低传代D3、D2和D52病毒株的UL144基因均全长531 bp.通过blast分析,从GenBank中找到了10株HCMV病毒株的UL144与D3、D2和D52的UL144基因具有较高的同源性,经过序列的比对,发现UL144基因DNA序列比较保守,只在4处有变异,且变异均为碱基替换,无插入或缺失,编码蛋白由176个氨基酸残基组成,氨基酸序列也比较保守,各分离株中变异率为1.1%;HCMV UL144编码蛋白翻译后修饰位点在所有分离株中均高度保守;所有分离株UL144蛋白的等电点均为8.97.结论 广州地区临床低传代分离株HCMV UL144基因DNA及其编码产物的氨基酸序列是比较保守的,但仍存在一定的多态性.提示UL144基因在先天性感染中可能具有重要作用.

abstractsObjective To investigate the polymorphism of human cytomegalovirus (HCMV) UL144 gene of the low passage clinical isolates in Guangzhou and explore the role of UL144 gene in HCMV pathogenicity. Methods The clinical isolates of HCMV were obtained from the urine sample collected from those infants with intra-uterus HCMV infection in Guangzhou. The virus genome DNA was extracted. According to the genome sequence of Toledo, primers for UL144 gene were designed and used to amplify the complete open reading frames (ORF) of the UL144 gene in our 3 different clinical isolates. These ORFs of the UL144 gene were cloned into pMD18-T vector and their sequences were confirmed by sequencing. Bioinformatics methods were used subsequently to analyze the polymorphisms of these genes in different stains. Results Three HCMV low passage clinical isolates were successfully isolated, named D2, D3 and D52. As shown by PCR, all of these three strains contained UL144 ORF region. Three complete ORFs were amplified in total and their sequences were submitted to GenBank (Accession No.: DQ180368, DQ180382 and DQ180355). In D2, D3 and D52 isolates, their UL144 ORFs consisted of 531 nucleotides. DNA sequences were quite conservative,all variability were base substitution, and the amino acid sequences were high conservative, the rate of amino acid variability was 1.1%. There were no additional or deleted sites of posttranslational modification of UL144 protein in all clinical isolates. There were some differences in the secondary structure among different isolates. The isoelectric point of UL144 protein of all clinical isolates was 8.97. Conclusions All DNA and deduced amino acid sequences of UL144 gene share great similarity among Guangzhou HCMV clinical strains regardless of their polymorphism. It implies that maybe UL144 gene plays an important role in congenital infection.